Satoshi Kawata
1,2
, Katsumasa Fujita
1
, and Prabhat Verma
1
1 Dept. of Applied Physics, Osaka University, Osaka, Japan
2
RIKEN, Wako city, Saitama, Japan
When one thinks about the molecular and cellular imaging, particularly, in biomedical sciences,
Raman spectroscopy always turns out to be advantageous over the other common techniques, because it is not only nondestructive, but also it does not need any staining of molecules, which guarantees preservation of the physiological activities of the sample molecules. When combined with microscopy, Raman spectroscopy takes advantage of the rich information available from the molecular vibrations, and helps providing high quality images constructed from the strength of vibrational modes. One problem in obtaining Raman image is the long time involved in the imaging process, which is due to the inherent weak signal of Raman scattering. We have come over this problem by developing a technique of slit-scanning confocal Raman microscopy. Apart from an improved acquisition rate, this new technique is based on collecting Raman signal from multiple points of the sample simultaneously using a line-shaped illumination, which is scanned over the sample in the direction perpendicular to the illumination line, providing a two-dimensional x-y-image. At the same time, the sample can also be moved in the z-direction to construct a 3-D image. This method is particularly suitable for fast and high quality 3-D cellular imaging.
The spatial resolution in confocal Raman microscopy is, however, restricted by the diffraction limits of the probing light, which is a few hundred nanometers for visible light, is not small enough to image nano-sized biomolecules and cells. This problem can be overcome by combining the near-field techniques with confocal Raman microscopy. In our previous work [1-3], we have shown that Raman scattering combined with near-field microscopy, i.e.
, tip-enhanced Raman spectroscopy
(TERS), provides super spatial resolution far beyond the diffraction limits of the probing light, along with an enhanced scattering efficiency. This is due to the nano-sized evanescent field created in close proximity of the apex of a nanometallic tip, which enhances the scattering from the sample molecules directly under the tip apex. Several nanomaterials, such as carbon nanotubes, carbon 60, and dye molecules, as well as some biomedical samples, such as DNA base molecules, protein molecules and cells have been successfully imaged with a spatial resolution down to about 25 nm.
The image quality, such as the contrast and the resolution, can be further improved by invoking higher-order scattering effects in Raman scattering process because it provides better confinement of light field than the linear scattering due to the nonlinear effects, and better contrast due to the suppression of the luminescence background. This has been shown by utilizing the near-field effects in coherent anti-Stoke Raman scattering (CARS), a thirdorder nonlinear scattering process. This combined imaging technique, known as the tipenhanced CARS, has been applied to image adenine molecules [4], and a high quality image with a spatial resolution of 15 nm was obtained. This technique is proved to be very successful for high quality imaging, particularly, of biomolecules.
[1] N. Hayazawa, et al., Chem. Phys. Lett. 376, 174 (2003).
[2] H. Watanabe, et al., Phys. Rev. B 69, 155418 (2004).
[3] P. Verma, et al., Phys. Rev. B 73, 045416 (2006).
[4] T. Ichimura, et al., Phys. Rev. Lett. 92, 220801 (2004).